Nectin-1 is a cell adhesion molecule localized the puncta adherentia junctions in synapses. Nectin-1 associates with other proteins, which collectively participate in the formation of neuronal synapses. We hypothesize that nectin-1-undergoes a regulated multi-step set of endoproteolytic cleavage events by several sheddases in an activity-dependent manner and that these events regulate synaptogenesis and contribute to synaptic plasticity. Our preliminary data indicate that nectin-1 undergoes ectodomain shedding by at least two sheddases that result in the production of two C-terminal fragments (CTFs). These CTFs are further cleaved intramembraneously by y-secretase and liberate the NE-ICD from the plasma membrane. The released NEICD translocates into the nucleus and where we postulate it induces gene expression. In Specific Aim 1, we will determine the secretase cleavage sites of nectin-1 by immunoaffinity purification, followed by Edman degradation sequencing. We will also investigate which genes are regulated by NE-ICD in hippocampal neurons by CodeLink Bioarrays. Then, using quantitative RT-PCR, ICC, and Western blotting we will confirm differentially expressed genes in neurons. The molecules that interact with NE-ICD will be identified by a yeast two-hybrid screen. Once interactors are identified, their biological function will be assayed by cellular and molecular approaches. In Specific Aim 2, we will investigate the biological role of BACE1 in nectin-1 processing. Initial experiments indicate that BACE1 associates with and participates in the shedding of nectin-1. We will investigate how disruption of nectin-1 shedding, through loss of BACE1 function, affects synapse formation and synaptic plasticity by ICC, Western blotting and-vesicle recycling assays. Our preliminary data indicate that two nectin-1 point mutations, T310A and Y311A, are refractory to BACE1 cleavage and can /ra".y-dominantly interfere with processing of endogenous nectin-1. We will examine how these point mutants affect the synapse formation and synaptic function by transduction of hippocampal neurons and in vivo adult hippocampus with recombinant adeno-associated viral vectors. We will quantitatively measure the changes in synaptic markers, synapse morphology, and size by ICC, live cell imaging and synaptic activity. Subsequently, we examine whether expression of trans-dominant nectin-1 mutants will affect hippocampal dependent learning